Electrophoresis gel cassette and comb

ABSTRACT

Provided herein is an apparatus for gel electrophoresis comprising a cassette and a comb having at least one wedge-shaped tooth.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Divisional of U.S. application Ser. No.14/158,696 filed Jan. 17, 2014, which is a continuation and claims theright of priority under 35 U.S.C. 120 to U.S. application Ser. No.12/862,460 filed Aug. 24, 2010 (now abandoned), which claims a benefitunder 35 U.S.C. § 119(e) from earlier filed U.S. ProvisionalApplications No. 61/237,287 filed Aug. 26, 2009, U.S. ProvisionalApplications No. 61/237,195 filed Aug. 26, 2009, U.S. ProvisionalApplications No. 61/236,795 filed Aug. 25, 2009, and U.S. ProvisionalApplications No. 61/236,293 filed Aug. 24, 2009, all of which arecommonly owned with the present application, and all of which are herebyexpressly incorporated by reference in their entirety as though fullyset forth herein.

FIELD

The present invention relates to gel consumables.

BACKGROUND

Gel electrophoresis is a common procedure for the separation ofbiological molecules, such as DNA, RNA, polypeptides and proteins. Ingel electrophoresis, the molecules are separated into bands according tothe rate at which an imposed electric field causes them to migratethrough a filtering gel.

The basic unit used in this technique consists of a gel enclosed in aglass tube or sandwiched as a slab between glass or plastic plates. Gelshave an open molecular network structure, defining pores which aresaturated with an electrically conductive buffered solution. These poresare large enough to admit passage of the migrating macromoleculesthrough the gel.

The gel is placed in a chamber in contact with buffer solutions whichmake electrical contact between the gel and the cathode or anode of anelectrical power supply. A sample containing the macromolecules and atracking dye is placed on top of the gel. An electric potential isapplied to the gel causing the sample macromolecules and tracking dye tomigrate toward the bottom of the gel. The electrophoresis is halted justbefore the tracking dye reaches the end of the gel. The locations of thebands of separated macromolecules are then determined. By comparing thedistance moved by particular bands in comparison to the tracking dye andmacromolecules of known size, the size of other macromolecules can bedetermined.

Polyacrylamide gels are commonly used for electrophoresis. Other gelssuitable for electrophoresis include agarose gels and starch gels.Polyacrylamide gel electrophoresis or PAGE is popular because the gelsare optically transparent, electrically neutral and can be made with arange of pore sizes.

Methods of making PAGE gels are well known. See B. Hames and D.Rickwood, Gel Electrophoresis of Proteins (2d ed. Oxford UniversityPress, 1990); A. Andrews, Electrophoresis (2nd ed. Oxford UniversityPress, 1986). In general, stock solutions containing acrylamide monomer,a crosslinker such as bisacrylamide, gel buffers and modifying agentssuch as sodium dodecyl sulphate (“SDS”) are prepared. These stocksolutions can be stored until a gel is needed. To manufacture a gel, thestock solutions are mixed with water in proportions according to thefinal desired concentrations of the various constituents.

Glass has typically been used to make molds for electrophoresis gels.However, glass suffers from the disadvantage that it is fragile,difficult to form into particular shapes and expensive. It is easier andmore economical to form gel molds from plastic materials by processessuch as injection molding. However, using plastic molds for castingelectrophoresis gels may contribute to decreased resolution of theseparated macromolecule bands. Decreased resolution of the macromoleculebands may be caused by macromolecules moving faster on the surface ofthe gel in contact with the mold than in the interior of the gel. Thisvariation in migration rates between the surface of the gel and theinterior lead may lead to smearing of the macromolecule bands.

In addition to the decreased resolution that may occur in a gel, anotherdisadvantage of current methods for creating and performing gels, suchas SDS-PAGE gels, is that the use of the gel is limited by the amount ofsample that may be loaded into the gel. It may be desirable to have theability to apply greater volumes of samples to the gels. Therefore itmay be desirable to produce gels of greater thickness or to produce gelswith wells with an increased well-volume proportional to the increasedthickness of the gel. However, this may lead to gels that requireproportionately greater current for a given field strength. Greatercurrent for a given field strength may lead to greater heat build up inthe gels which in turn may lead to decreased resolution and performance.Another problem that may arise with thicker gels is that protein bandsmay transfer less efficiently in down stream applications, such aswestern blotting.

One solution for increasing sample volume in a gel may be to increasethe depth of the wells of a particular width and gel thickness. However,the increased volumes may produce increased sample heights above thegel, leading to thicker protein band starting zones, and lowerresolution. If the stacking gel height is proportionately increased,then the resolving gel length will be proportionately decreased, alsocontributing to reduced resolution in a given gel cassette.

Therefore, it would be beneficial to develop a gel cassette that wouldenable producing a gel of a particular thickness having a well capableof holding a sample volume that is at least double the well volume ofthe current methods, while maintaining the same sample height above thegel as a standard well of particular width and thickness.

SUMMARY

Provided herein is an apparatus for gel electrophoresis comprising: agel cassette; and a comb having at least one wedge-shaped tooth. In someembodiments, the gel cassette may include a retainer plate and a dividerplate. The gel cassette includes a lip extending from the exteriorsurface of at least one of the divider plate or the retainer plate. Insome embodiments, the wedge-shaped tooth has a volume of at least 40 uL.In some embodiments, the cassette has at least one interior surface thatmay be coated with a coating. In some embodiments, the coating is anoxygen interference coating, such as, for example, at least one of SiOor SiO₂. In some embodiments the comb further comprises a recessed areaon the comb wherein the recessed area is configured to prevent theformation of a skin in a gel during gel formation.

Further provided herein is an apparatus for gel electrophoresiscomprising a comb having wedge-shaped teeth configured to create asample well in a 1 mm gel, wherein the sample well size is at least 10%larger than a gel having a sample well created by a standard comb.

Provided herein is an apparatus for gel electrophoresis comprising acomb having wedge-shaped teeth configured to create a sample well in a 1mm gel, wherein the sample well size is at least 100% larger than a gelhaving a sample well created by a standard comb. In some embodiments,the sample well created by a standard mini gel ten teeth comb has avolume of 37.5 μL.

Further provided herein is an apparatus for gel electrophoresiscomprising a ten teeth mini gel comb having wedge-shaped teethconfigured to create a sample well in a 1 mm gel, wherein the samplewell has a volume of at least 70 μL.

An apparatus for gel electrophoresis comprising a comb havingwedge-shaped teeth configured to create sample wells configured toreceive at least 10% more sample than a standard gel sample well. Insome embodiments, the apparatus is configured to create a gel havingsharp bands. In some embodiments, the apparatus is configured to createa gel having high resolution.

Provided herein is an apparatus for gel electrophoresis comprising: agel cassette having a lip extending at an angle from at least oneexterior surface of the cassette; and a comb having at least onewedge-shaped tooth.

Further provided herein is an apparatus for gel electrophoresiscomprising a gel cassette having a cavity; and a comb; and at least onetooth located on the comb, the at least one tooth having at least oneside that is slanted, wherein the at least one tooth is located at least0.335 inches from the edge of the cavity.

Provided herein is an apparatus for gel electrophoresis comprising acomb having wedge-shaped teeth configured to create sample wellsconfigured to receive at least 10% more sample than a standard gelsample well. In some embodiments, the device may be configured to createa gel that isolates the desired component as a sharp band or a highresolution band or both.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the novel features of the invention are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of the present invention will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the invention areutilized, and the accompanying drawings of which:

FIG. 1 shows an embodiment of an assembled gel cassette according tovarious embodiments of the present teachings;

FIG. 2A shows an embodiment of a divider plate according to variousembodiments of the present teachings;

FIG. 2B shows an embodiment of a retainer plate according to variousembodiments of the present teachings;

FIG. 3 shows an embodiment of an assembled gel cassette according tovarious embodiments of the present teachings;

FIG. 4 shows an embodiment of a cassette and an embodiment of a combaccording to various embodiments of the present teachings;

FIGS. 5A-5C show various views of an embodiment of a comb;

FIGS. 6A & 6B show images of gels;

FIG. 7 shows a comparison of results from two different gel types;

FIGS. 8A-8C are a side view, a cross-sectional view, and a bottom view,respectively, of a single well comb according to various embodiments ofthe present teachings;

FIGS. 9A-9C are a side view, a cross-sectional view, and a bottom view,respectively, of a nine well comb according to various embodiments ofthe present teachings;

FIGS. 10A-10C are a side view, a cross-sectional view, and a bottomview, respectively, of a ten well comb according to various embodimentsof the present teachings;

FIGS. 11A-11C are a side view, a cross-sectional view, and a bottomview, respectively, of a twelve well comb according to variousembodiments of the present teachings;

FIGS. 12A-12C are a side view, a cross-sectional view, and a bottomview, respectively, of a fifteen well comb according to variousembodiments of the present teachings; and

FIGS. 13A-13C are a side view, a cross-sectional view, and a bottomview, respectively, of a seventeen well comb according to variousembodiments of the present teachings.

DETAILED DESCRIPTION

Provided herein is an apparatus for gel electrophoresis comprising a gelcassette and a comb having at least one wedge-shaped tooth. The gelcassette may include a retainer plate and a divider plate. The platesmay be formed by a process such as injection molding. Any suitableplastic may be used for forming the plates, such as for example purposesonly polymers such as polyethylene terephthalate, polyvinyl chloride,polymethyl methacrylate, polystyrene, polyethylene, polymethylpolypropylene, and cellulose acetates or various co-polymers. In someembodiments, the plastic may be a transparent plastic so that the gelmay be viewed inside the assembled cassette. In some embodiments, thecassette may be constructed from glass plates, or may be constructedfrom one glass plate and one plastic plate. In some embodiments, thedevice may be configured to create precast gels that lay flat on varioussurfaces. The ability to create precast gels that lay flat may bebeneficial for product shipping and storage, as well as during routinehandling by the user. Additionally, the gel cassette may lay flat inauto-loading machinery during gel manufacturing.

In some embodiments, the divider plate has a series of features, bumps,or posts located along the length of either the sides of the plate,along the bottom length of the plate or both along the sides and bottomof the plate. The posts may function as energy directors during theassembly process to create a uniform seal around the plate. In someembodiments, a slot may be located at the bottom portion of the dividerplate, and in some embodiments, may or may not extend through thethickness of the plate. The slot may expose the gel inside the cassetteto a buffer, such as an anode buffer, to complete or assist incompleting an electrical path circuit during electrophoresis. Duringformation of the gel, the slot may be sealed with a sealant, such astape, epoxy, polymers, conductive polymers or any other suitable sealantto prevent the gel material from escaping the assembled cassette priorto setting of the gel. In some embodiments, the divider plate mayinclude a ridge that together with the spacer on the retainer platecreates the gel cavity. In some embodiments, the sealant may be presenton the divider plate prior to assembling of the cassette, or the sealantmay be placed over the slot on the assembled cassette by the user priorto filling the assembled cassette with a gel material. After the gel hasbe formed, the sealant may be removed if a non-conductive sealant isused, so to expose the gel to a buffer to complete or assist incompleting an electrical path circuit. At the top of the divider plate,the top surface may include a grip or a curved surface to facilitatehandling of the assembled cassette.

In some embodiments, at least one of the plates, for example, theretainer plate may have a spacer located on its interior surface. Thespacer may be any suitable shaped spacer, for example, a generallyu-shaped raised spacer located on the interior surface. In someembodiments, the spacer may be located on the interior surface of oneplate and contact the interior surface of the other plate. In someembodiments, a spacer may be located on the interior surface of bothplates and the two spacers may contact each other when the cassette isformed. The spacer or spacers may further serve to help maintain thespace between the divider plate and the retainer plate when the platesare joined to form a cassette. When the retainer plate and the dividerplate are joined together, the interior surfaces of the plates form aslab shaped cavity which may be sealed by the raised spacer. In someembodiments, the ridge located on the divider forms the seal between thedivider plate and the retainer plate. In some embodiments, both thespacer and the ridge seal the gel cavity. In some embodiments, theretainer plate and the divider plate may be fabricated as a single pieceof plastic. In some embodiments, the retainer plate and the dividerplate may be two separate parts that are connected together. The twoseparate parts may be connected together by any suitable mechanism forattaching the plates together including, for example purposes only,glue, pressure fitting, welding, adhesive, clamps, clips, or any othersuitable mechanism. The thickness of the cavity may be determined by theheight of the spacer. In some embodiments, the cavity formed by thedivider plate and the retainer plate is capable of holding gel materialthat may form a gel about 1 mm thick.

In some embodiments, a lip extends at some angle away from the exteriorsurface of one of the plates to form an opening. Gel material may enterinto the cassette through the opening. Additionally, a structure forforming sample wells in the gel may be introduced to the gel materialthrough the opening. In some embodiments, the lip may extend from theexterior surface of one of the plates at an angle, for example at abouta 70 degree angle from a plane perpendicular to the exterior surface ofthe plate, or 20 degrees from surface of the plate. In some embodiments,the angle may be between about 15 degrees and about 25 degrees, betweenabout 10 degrees and 30 degrees, between about 5 degrees and 35 degrees,or any other suitable angle. In some embodiments, the lip may extendfrom the exterior surface of the plate by less than about 45 degrees.Projections may be located on the exterior surface of the retainer plateto support the lip. In some embodiments, the projections may alsofacilitate stacking the welded cassettes in a magazine, aiding in themanufacturing process. In some embodiments, an assembled cassette mayhave at least one lip on at least one side to create an opening that maybe able to accommodate a comb for creating wells in the gel where thecomb is wedge-shaped, such as any of the combs described herein. In someembodiments, a lip may be located on both sides of the opening, creatinga v-shaped opening to accommodate v-shaped teeth on a comb. In someembodiments, the opening of the cassette may have an opening that isflat on both sides, but is wide enough to accommodate wedge shaped orv-shaped teeth on a comb without the need for a lip extending from oneof the surfaces of the plate.

In some embodiments of the device, the cassette has at least oneinterior surface which may be coated with a coating. The coating may bean oxygen interference coating (a coating that forms an oxygen barrierthat reduces diffusion from the surface of the plastic plates into thegel to prevent significant local variations in the rate of oxygendiffusion) such as, for example purposes only, a coating comprisingSiO_(x), SiO, or SiO₂. In some embodiments the coating may besubstantially transparent. The interior surfaces maybe coated manuallyor mechanically and in some embodiments where two separate plates areformed into the cassette, the coating may be performed before or afterjoining the plates into the cassette. In some embodiments, the interiorsurfaces of the cassette may be coated under vacuum using evaporation orvapor deposition, sputter deposition, chemical vapor deposition,plasma-enhanced chemical vapor deposition, or any combination thereof.In some embodiments, the coating deposited may be of a thickness lessthan about 5000 Angstroms in thickness.

During preparation of a gel, the slot in the divider plate may besealed. The slot may be sealed by any suitable sealant including tape,epoxy, glue, polymer compound, or any other suitable sealant. Thesealant may or may not be a conductive sealant. In some embodiments, thesealant may be a permanent sealant or a removable sealant. Once the slothas been sealed, the cassette may be held vertically and gel-formingmixture is poured through the opening into the cavity formed between theretainer plate and the divider plate. The gel mixture may be in contactwith the interior surfaces of the divider plate and the retainer plate.

Further provided herein is an apparatus for gel electrophoresiscomprising a comb having wedge-shaped teeth configured to create asample well in a 1 mm gel, wherein the sample well size is at leastabout 10% larger than a gel having a sample well created by a standardcomb. In some embodiments, the sample well size created by a standardcomb in 37.5 μL. In some embodiments, the sample well size created bythe wedge well comb may be at least 41 μL. In some embodiments, thesample well size created by the wedge well may be at least about 50 μL,at least 60 μL, at least about 70 μL, at least about 75 μL.

Provided herein is an apparatus for gel electrophoresis comprising acomb having wedge-shaped teeth configured to create sample wellsconfigured to receive at least 10% more sample than a standard gelsample well. Increased sample size may allow for more sample to beloaded into the gel without causing damage to the gel and yet stillproduce sharp, high resolution bands. Additionally, a large sample wellopening may facilitate loading samples with standard pipette tipsinstead of special drawn-out gel loading pipette tips.

Once the gel material for forming a gel has been poured into the openingof an assembled cassette, a comb may be inserted into the opening of theassembled cassette and into the gel material. The depth of insertion ofthe comb may be limited by the edges of the comb and the shoulders ofthe opening located on the cassette. In some embodiments, the depth ofinsertion of the comb may be further limited by the comb resting on thefront edge of the lip. Once the gel has been cast, the comb may then beremoved leaving a void in the gel at the position of each tooth. Eachvoid forms a sample well having a sample volume.

In some embodiments, the comb may have wedge-shaped teeth. A comb havingwedge-shaped teeth may be capable of creating wedge-shaped wells in thegel. In some embodiments, the wedge-shaped teeth have flat surfaces atthe tips of the teeth. The flat bottom surface of the teeth createssample wells having flat bottoms. The flat bottom of the sample wellbecomes the leading edge of the stacked sample bands which mayultimately resolve into bands that are as flat and similar to bandscreated by standard combs. The wedge-shaped wells created by insertionof the comb with wedge shaped teeth into the gel material may provide alarger opening to the well at the top of the sample well and therebycreate easier access to the wells using a standard pipette tip on asingle pipette or on a multi-channel pipette. Additionally, thewedge-shaped wells permit a greater amount of sample to be loaded into athinner gel (for example, approximately 1 mm thick gels). The comb maybe used with any of the cassettes described herein previously, or may beused with any other suitable cassette for creating a gel.

The wedge-shaped wells in the gel created by the wedge shaped teeth maybe of a volume at least 10% greater than the volume of the wells createdusing a standard comb. In some embodiments, a standard comb may createwells having a volume of about 37.5 μL. In some embodiments, the wedgeshaped wells may have a volume that is at least 20% greater, at least50% greater, at least 100% greater, at least 150% greater, at least 175%greater, at least 200% greater, at least 250% greater than a wellcreated using a standard comb. In some embodiments, the sample well sizecreated by the wedge well comb may be at least 41 μL. In someembodiments, the sample well size created by the wedge well may be atleast about 45 μL, at least about 55 μL, at least 60 μL, at least about70 μL, at least about 75 μL, at least about 90 μL, at least about 100μL, at least about 110 μL, at least about 130 μL.

Additionally, the wedge-shaped wells may allow for loading an increasedamount of sample while maintaining or improving band sharpness andresolution during electrophoresis while reducing edge effects. The wedgeshaped design of the wells formed by the teeth of a wedge-tooth comballows more sample to be loaded into a well, while keeping the sampleaway from the sides of the gel. Sample that is located toward the endsof the gel may not form as sharp bands or bands having high resolution.Positioning of the wells at such locations reduces the chance that thebands formed in the gel will turn up at the ends of the band or “smile”or turn down at the edges of the band or “frown.” Additionally, thewedge-shaped wells created using the apparatus described herein have agreater cross sectional area at the top of the sample well as comparedto the bottom of the sample well. The increase in cross sectional areacreates a lower voltage drop at any given constant voltage, current orwattage at the top of the sample well compared to the bottom of thesample well. The lower voltage drop, or field strength, in this area maylead to better sample stacking than in the thinner sections of the welland/or gel, or in gels with wells of the same thickness through out thesample loading volume.

In some embodiments, the gel cassettes are about 10 cm by 10 cm formini-gel cassettes. The gels housed in the gel cassettes are about 7 cmin length (the running length) by about 8 cm wide. The gel cassettes andgels run in the gel cassettes may be any suitable length. In someembodiments, the gel cassette width may be large enough to reduce edgeeffects in gels having at least 10 wells. In some embodiments, theassembled cassette may include a gap that runs around the outerperimeter of the cassette. The gap may facilitate the opening of thecassette for gel retrieval. In some embodiments, the gel cassette may beopened with a gel knife or may be opened by hand.

In some embodiments, the comb may be a ten-tooth comb, that is, a combused for forming 10 sample wells in a gel. In some embodiments, the combmay be at least a single tooth comb, at least a 2-tooth comb, at least afive-tooth comb, at least a seven-tooth comb, at least a 9-tooth comb,at least a 10-tooth comb, at least a twelve-tooth comb, at least a15-tooth comb, at least a 17-tooth comb, at least a 20-tooth comb, orany other comb having a suitable number of teeth. In some embodiments,the comb may be molded from any suitable plastic including, but notlimited to polymers such as polyethylene terephthalate, polyvinylchloride, polymethyl methacrylate, polystyrene, polyethylene, polymethylpolypropylene, cellulose acetates, co-polymers, polycarbonate, or anyother suitable material. In some embodiments, the dimensions of theteeth may be uniform along the length of the comb or the teeth may havevarying dimensions along the length of the comb. The teeth may or maynot be the same shape as the wedge shaped opening of the cassette.

In some embodiments, the edge of the comb opposite to the teeth mayserve as a handle that may facilitate the insertion and removal of thecomb from the cassette. In some embodiments, the edge may be curved. Thecomb may further include a back surface and a front surface. In someembodiments, the front and back surfaces are solid. In some embodiments,the front surface of the comb has a recessed space which facilitatesgripping the comb by the user to facilitate the insertion of the combinto the cassette as well as to remove the comb from the cassette oncethe gel and wells have been formed.

I. CASSETTE

FIG. 1 shows an embodiment of an assembled gel cassette 100. Theassembled gel cassette 100 comprises two plastic plates, a retainingplate 104 and a divider plate 102. The plates may be formed by a processsuch as injection molding. Any suitable plastic may be used for formingthe plates 102, 104. Suitable plastics for forming electrophoresis gelmolds include polymers such as polyethylene terephthalate, polyvinylchloride, polymethyl methacrylate, polystyrene, polyethylene, polymethylpolypropylene, and cellulose acetates or various co-polymers. In someembodiments, the plastic may be a transparent plastic so that the gelmay be viewed inside the assembled cassette 100. In some embodiments,the cassette 100 may be constructed from glass plates, or may beconstructed from one glass plate and one plastic plate.

Referring to FIG. 2A, one embodiment of a divider plate 202 is shownhaving an interior surface 206. In some embodiments, the divider platehas a series of features, bumps, or posts 240 located along the lengthof the sides of the plate 204 and along the bottom length of the plate.The posts 240 may protrude from the divider plate 202 and uponattachment of the divider 202 to the retainer plate (204 in FIG. 2B) theposts 240 may connect with the retainer plate. As shown in FIG. 2A, thedivider plate 202 may additionally include a ridge 241 that runs downthe sides and across the bottom of the divider plate 202. The ridge maybe used to seal the gel cavity on the assembled cassette. In someembodiments, a slot 214 may be located at the bottom portion of thedivider plate 202, and in some embodiments, may or may not extendthrough the thickness of the plate 204. The slot 214 may expose the gelinside the cassette to a buffer, such as an anode buffer, to complete orassist in completing an electrical path circuit during electrophoresis.On the back surface of the plate a groove 242 may or may not be present.Additionally, cut-outs 244, 246 may be located on the back surface 248of the plate to facilitate removal of any sealant blocking the slot. Atthe top of the divider plate 202, the top surface 252 may include a gripor a curved surface, as shown in FIG. 2A, to facilitate handling of theassembled cassette.

FIG. 2B shows an embodiment of a retainer plate 204 having an interiorsurface 208. In some embodiments, the retainer plate 204 may have agenerally u-shaped raised spacer 210 located on the interior surface 208which may contact the divider plate 202. The u-shaped spacer 210 mayfurther serve to help dictate the spacing between or to maintain thespace between the retainer plate 204 and the divider plate 202 when theplates are joined to form a cassette. When the divider plate 202 and theretainer plate 204 are joined together, the interior surfaces 206, 208of the plates form a slab shaped cavity which may be bounded by theraised spacer 210. During assembly, either the spacer 210, the ridge 241on the divider plate 204 (in FIG. 2B), or the spacer 210 and ridge 241may seal the cavity. The thickness of the cavity is determined by theheight of the u-shaped spacer 210. In some embodiments, the cavityformed by the divider plate 202 and the retainer plate 204 is capable ofcontaining and confining gel material that may form a gel 1 mm thick.

In some embodiments, a lip 216 extends at some angle away from theexterior surface of one of the plates to form the opening, for examplefrom the exterior surface 217 of the retainer plate 204 as shown in FIG.2B. The lip further includes a first and second shoulder 236, 238,respectively, and a front edge 258. Projections 254, 256 may be locatedon the exterior surface 217 of the retainer plate to support the lip216. In some embodiments, the projections 254, 256 may also facilitatestacking the welded cassettes in a magazine, aiding in the manufacturingprocess. In some embodiments, an assembled cassette may have at leastone lip 216 on at least one side to create an opening that may be ableto accommodate a comb for creating wells in the gel where the comb iswedge-shaped, such as any of the combs described herein. In someembodiments, a lip may be located on both sides of the opening, creatinga v-shaped opening.

An assembled cassette 300 formed from a retainer plate 304 and a dividerplate 302 is shown in FIG. 3. An opening 312 created by joining of theretainer plate 304 and the divider plate 302 may be located at the topof the assembled cassette 300. In some embodiments, the opening 312formed by the retainer plate 304 and the divider plate 302 is flat onone side while the other side of the opening is configured so that theopening of the assembled cassette may receive a comb having wedge shapedteeth or v-shaped teeth.

In some embodiments, the retainer plate 304 and the divider plate 302may be fabricated as a single piece of plastic. In some embodiments, theretainer plate 304 and the divider plate 302 may be two separate partsthat are connected together. The two separate parts may be connectedtogether by any suitable mechanism for attaching the plates togetherincluding, for example purposes only, glue, pressure fitting, welding,adhesive, clamps, clips, or any other suitable mechanism.

During preparation of a gel, the slot 314 in the divider plate 302 maybe sealed. The slot 314 may be sealed by any suitable sealant includingtape, epoxy, glue, polymer compound, or any other suitable sealant. Thesealant may or may not be a conductive sealant. In some embodiments, thesealant may be a permanent sealant or a removable sealant. Once the slot314 has been sealed, the cassette 300 may be held vertically andgel-forming mixture is poured through the opening 312 into the cavity362 formed between the retainer plate 304 and the divider plate 302.After the gel has been cast, an electric circuit may be formed eitherthrough a conductive sealant material, or alternatively, the sealant canbe removed from the exterior of the cassette, thereby exposing the gelinside the cassette to a buffer solution and thereby completing theelectric circuit path.

The gel mixture may be in contact with the interior surfaces 306, 308 ofthe divider plate 302 and the retainer plate 304, respectively. In someembodiments, the interior surfaces 306, 308 of the divider plate 302 andthe retainer plate 304, respectively, may be coated. In someembodiments, the one or both of the interior surfaces 306, 308 of theplates may be coated with an oxygen interference coating such asSiO_(x), for example, SiO or SiO₂, or any combination thereof or anyother suitable coating, which may form an oxygen barrier that reducesdiffusion from the surface of the plastic plates into the gel to preventsignificant local variations in the rate of oxygen diffusion. Methodsfor coating plastic gel molds can be found in U.S. Pat. No. 5,685,967,which is incorporated herein by reference in its entirety. In someembodiments the coating may be substantially transparent. The interiorsurfaces 306, 308 may be coated manually or mechanically and in someembodiments where the cassette is formed from two separate plates, thecoating may be performed before or after joining the plates into thecassette. In some embodiments, the interior surfaces 306, 308 of thecassette may be coated under vacuum using evaporation or vapordeposition, sputter deposition, chemical vapor deposition,plasma-enhanced chemical vapor deposition, or any combination thereof.In some embodiments, the coating deposited may be of a thickness lessthan about 5000 Angstroms in thickness.

In some embodiments, the gel cassettes are about 10 cm by 10 cm formini-gel cassettes. The gels housed in the gel cassettes are about 7 cmin length (the running length) by about 8 cm wide. The gel cassettes andgels run in the gel cassettes may be any suitable length. In someembodiments, the assembled cassette may include a gap 360 that runsaround the outer perimeter of the cassette 300. The gap 360 mayfacilitate the opening of the cassette for gel retrieval.

After the gel has been loaded with sample, the gel cassette containingthe samples in the wells may be placed into an electrophoresis devicethat creates a liquid-tight seal between the external margins of theretainer and the divider plates. After being placed in theelectrophoresis device, the electrode buffer is placed in the anode tank(formed behind the sealed divider) and the cathode tank (formed behindthe sealed retainer). When a voltage is applied to the electrophoresisdevice anions flow from the cathode tank down through the sample wellsand gel into the anode tank, while cations flow into the bottom, andthrough, the gel from the anode tank. The gel cassette may be used withany suitable electrophoresis device or system, including the systemdescribed in U.S. Provisional Application Ser. No. 61/237,287, filedAug. 26, 2009, the contents of which are incorporated by reference intheir entirety.

II. COMB

As shown in FIG. 4, once the gel material has been poured into theopening of an assembled cassette 400, a comb 420 may be inserted intothe opening of the assembled cassette, so that the teeth of the combcontact the top portion of the gel material. The depth of insertion ofthe comb 420 may be limited by the edges 432, 434 of the comb 420 andthe shoulders 436, 438 of the opening located on the cassette 400. Insome embodiments, the depth of insertion of the comb 420 may be furtherlimited by the comb 420 resting on the front edge 458 of the lip 416.Once the gel has been cast, the comb may then be removed leaving a voidat the position of each tooth. The voids formed by the teeth of the combare the sample wells in the gel.

FIGS. 5A-5C show different views of an embodiment of a comb 520 to beused with a gel cassette provided herein. In some embodiments, the comb520 may have wedge-shaped teeth 522, as shown in FIGS. 5A-5C. A comb 520having wedge-shaped teeth 522 may be capable of creating wedge-shapedwells in the gel. The wedge-shaped wells created by insertion of thecomb with wedge shaped teeth into the gel material may provide a largeropening to the well at the top and thereby create easier access to thewells using a standard pipette tip on a single pipette or on amulti-channel pipette. Additionally, the wedge-shaped wells permit agreater amount of sample to be loaded into a thinner gel (for example,approximately 1 mm thick gels). The comb 520 may be used with any of thecassettes described herein previously, or may be used with any othersuitable cassette for creating a gel.

The wedge-shaped wells in the gel created by the wedge shaped teeth maybe of a volume at least 10% greater than the volume of the wells createdusing a standard comb. In some embodiments, the wedge shaped wells mayhave a volume that is at least 20% greater, at least 50% greater, atleast 100% greater, at least 150% greater, at least 175% greater, atleast 200% greater, at least 250% greater than a well created using astandard comb. Additionally, the wedge-shaped wells allow for loading anincreased amount of sample while maintaining or improving band sharpnessand resolution during electrophoresis.

In some embodiments, the comb 520 is a nine-tooth comb, as shown inFIGS. 5A & 5C. In some embodiments, the comb may be at least a singletooth comb, at least a 2-tooth comb, at least a five-tooth comb, atleast a seven-tooth comb, at least a 9-tooth comb, at least a ten-toothcomb, at least a twelve-tooth comb, at least a 15-tooth comb, at least a17-tooth comb, at least a 20-tooth comb, or any other comb having asuitable number of teeth. In some embodiments, the comb may be moldedfrom any suitable plastic including, but not limited to polymers such aspolyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate,polystyrene, polyethylene, polymethyl polypropylene, cellulose acetates,co-polymers, polycarbonate, or any other suitable material. In someembodiments, the dimensions of the teeth may be uniform along the lengthof the comb or the teeth may have varying dimensions along the length ofthe comb. The teeth 522 may or may not be the same shape as the wedgeshaped opening of the cassette.

In some embodiments, the edge 520 of the comb located opposite to theteeth 522 may be a curved edge 524. The curved edge 524 may facilitatethe insertion and removal of the comb 520 from the cassette. The comb520 may further include a back surface 526 and a front surface 528. Insome embodiments, the front 528 and back 526 surfaces are solid. Theback surface 526 of the comb 520 may be flush with the interior surface406 of the divider plate 402 (in FIG. 4), and the front surface of thecomb 520 may be align with the front edge 458 of the lip 416 of theretainer plate 404 (shown in FIG. 4). In some embodiments, the frontsurface 528 of the comb 520 has a recessed space 558 which inconjunction with the back surface 526 of the comb 520 facilitatesgripping the comb 520 to facilitate the insertion of the comb into thecassette by the user. The recessed space 558 may further assist in theremoval of the comb 520 from the cassette by the user, once the gel andwells have been formed. In some embodiments of the comb, the comb has arecessed area in the back of the comb where the comb contacts thecassette. In some embodiments, the recessed area helps to eliminatepolymerized gel formation in the sample wells of the electrophoresisgel. The recessed area helps prevent the formation of a “skin” in thesample wells during the gel casting process since the recessed areaeliminates geometrically induced capillary forces. Additionally, therecessed area helps to make loading the sample problem free bypreventing any gel material from being pushed into the sample well bythe pipette tip as the sample is loaded into the well.

As shown in FIGS. 5A & 5C, in some embodiments the teeth 522 on the comb520 may be fabricated such that the first and last well on the comb arelocated a suitable distance from the edges of the comb. By positioningthe teeth a suitable distance from the edges of the comb, a gel can becreated where the wells are located a suitable distance from the sidesof the gel thereby increasing the quality, resolution, and sharpness, ofthe bands. Such a location of the wells reduces the chance that thebands formed in the gel will turn up at the ends of the band or “smile”or turn down at the ends of the band or turn down at the edges of theband or “frown” or otherwise deviate from the horizontal straightnessand become more angled.

FIG. 5B shows a cross-sectional side view of comb 520 from FIGS. 5A and5C. As seen in FIG. 5B, the wedge-shaped teeth 522 taper along thelength of the tooth so that the well formed with the wedge comb 522 iswider at the top end of the gel and narrower or tapered at the bottomend of the well. The depth of the sample well formed in the gel is asdeep as the height of the angled portion of the tooth 522 from the tipto the edge 532 of the comb and is further defined by the space betweenthe angled-surface teeth.

In some embodiments, the bottom surface 534 of the wedge may be about0.04 cm in depth (as indicated by the arrows). In some embodiment, thebottom surface 534 may be between about 0.03 inch and about 0.05 inch.The top of the tooth 535 may be about 0.18 inch. In some embodiments,the top of the tooth 535 may be between about 0.15 and about 0.2 inch.At least one surface 533 of the wedge may extend from a plane parallelto the bottom surface of the tooth at an angle of 70 degrees. In someembodiments, the at least one surface 533 may extend from the bottomsurface plane at an angle of between about 45 degrees and about 90degrees.

FIGS. 8A-8C are a side view, a cross-sectional view, and a bottom view,respectively, of a single well comb according to various embodiments ofthe present teachings, and show various dimensions in inches, andangles, that can be used for such a configuration. FIG. 8B is across-sectional view taken along line A-A shown in FIG. 8A.

FIGS. 9A-9C are a side view, a cross-sectional view, and a bottom view,respectively, of a nine well comb according to various embodiments ofthe present teachings, and show various dimensions in inches, andangles, that can be used for such a configuration. FIG. 9B is across-sectional view taken along line A-A shown in FIG. 9A.

FIGS. 10A-10C are a side view, a cross-sectional view, and a bottomview, respectively, of a ten well comb according to various embodimentsof the present teachings, and show various dimensions in inches, andangles, that can be used for such a configuration. FIG. 10B is across-sectional view taken along line A-A shown in FIG. 10A.

FIGS. 11A-11C are a side view, a cross-sectional view, and a bottomview, respectively, of a twelve well comb according to variousembodiments of the present teachings, and show various dimensions ininches, and angles, that can be used for such a configuration. FIG. 11Bis a cross-sectional view taken along line A-A shown in FIG. 11A.

FIGS. 12A-12C are a side view, a cross-sectional view, and a bottomview, respectively, of a fifteen well comb according to variousembodiments of the present teachings, and show various dimensions ininches, and angles, that can be used for such a configuration. FIG. 12Bis a cross-sectional view taken along line A-A shown in FIG. 12A.

FIGS. 13A-13C are a side view, a cross-sectional view, and a bottomview, respectively, of a seventeen well comb according to variousembodiments of the present teachings, and show various dimensions ininches, and angles, that can be used for such a configuration. FIG. 13Bis a cross-sectional view taken along line A-A shown in FIG. 13A.

III. EXAMPLES

4-12% gels, as described in U.S. Provisional Application No. 61/236,293,filed Aug. 24, 2009, which is incorporated by reference in its entirety,were cast using a standard NOVEX® 1.5 mm Mini cassette and comb andusing a test 1.0 mm wedge-well cassette and comb described herein inFIGS. 1-4 and 10A-10C. The gel in the NOVEX® cassette was cast using astandard (flat) 1.5 nm, 10-well comb. The gel in the test cassette wascast using a 10-well wedge shaped comb. Five lanes of each the NOVEX®cassette gel and the test cassette gel were loaded with 5 μL of MARK 12™unstained standard diluted with NuPAGE® Sample Buffer to a final volumeof 37.5 μL. In each of the five lanes of each gel, 37.5 μL of dilutedstandard was loaded, so that the performance of the gels could beevaluated by comparing volume of standard loaded into the gels. Each gelwas run individually at 150 mA. The wedge well gel was run for 15 minand the 1.5 mm gel was run for 21 minutes. Upon completion of the runs,the gels were stained using SIMPLY BLUE SAFESTAIN, imaged on a flatbedscanner, and analyzed using NonLinear Dynamics Total Lab SoftwareVersion TL100. The software was used for the purpose of analyzing bandsharpness. The band sharpness was measured using the automatic functionfor the generation of peaks, where the software determines the peak'sstart and end points. The difference between the end point and startpoint of the peak correlates to the band sharpness and is reported inmillimeters. The results show that the bands on a 1.0 mm wedge well gel(FIG. 6A) are as sharp as the bands on a 1.5 mm standard mini gel (FIG.6B). In some embodiments, the sample may be stained by any suitablestain and staining procedure, including those described in U.S. patentapplication Ser. No. 12/122,607, filed May 16, 2008 and U.S. ProvisionalApplication No. 61/236,795, filed Aug. 25, 2009, which are bothincorporated herein by reference in their entireties.

FIG. 7 shows the comparison between the band sharpness using the 1.5 mmMini cassette gel and standard comb and the wedge-well cassette and combfor diluted MARK 12 standard. Although the MARK 12 standard contains 12bands, only the first ten bands were analyzed or compared in each lane.Some of the bands of the MARK 12 standard appear wavy on the 1.5 mm gel,particularly at the edge of the gel. The bands compared are as indicatedin FIGS. 6A & 6B. The sharpness of the band across all five wells (n=5)and standard deviation is indicated on the y-axis and the band positionalong the x-axis. FIG. 7 shows a generally increased performance usingthe test cassette versus using the NOVEX® cassette.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An apparatus for gel electrophoresis comprising:a gel cassette having a retainer plate and a divider plate; and a comb,comprising a recessed area on the back of the comb where the combcontacts the gel cassette and having at least one tooth configured tocreate a sample well, the tooth and the corresponding sample well havinga volume of at least 40 μl.
 2. The apparatus of claim 1, wherein thetooth and the corresponding sample well has a volume of at least 41 μL,at least about 45 μL, at least about 55 μL, at least 60 μL, at leastabout 70 μL, at least about 75 μL, at least about 90 μL, at least about100 μL, at least about 110 μL, or at least about 130 μL.
 3. Theapparatus of claim 1, wherein the sample well has a volume that is atleast 10% greater, at least 20% greater, at least 50% greater, at least100% greater, at least 150% greater, at least 175% greater, at least200% greater, or at least 250% greater than a well created using astandard comb, wherein the volume of sample wells created by using thestandard comb is about 37.5 μL.
 4. The apparatus of claim 1, wherein thegel cassette includes a lip extending from the exterior surface of atleast one of the divider plate or the retainer plate.
 5. The apparatusof claim 1, wherein the comb is a ten-tooth comb, a twelve-tooth comb, afifteen-tooth comb, a-seventeen tooth comb, or a twenty-tooth comb. 6.The apparatus of claim 1, wherein the comb is molded from a plasticcomprising: polyethylene terephthalate, polyvinyl chloride, polymethylmethacrylate, polystyrene, polyethylene, polymethyl polypropylene,cellulose acetates, copolymers, or polycarbonate.
 7. The apparatus ofclaim 1, further comprising a gel cassette with an opening into whichthe comb is insertable.
 8. The apparatus of claim 1, wherein the combcomprises a back surface that is flush with an interior surface of adivider plate of the cassette and a front surface that is aligned with afront edge of a lip of a retainer plate of the cassette.
 9. Theapparatus of claim 1, wherein the at least one tooth has a wedge-shape.10. The apparatus of claim 9, wherein the wedge-shaped teeth have atleast one surface extending from a plane parallel to the bottom surfaceof the teeth at angle of between 45 degrees and 90 degrees to the top ofthe teeth.
 11. The apparatus of claim 9, wherein the wedge-shaped teethhave flat surfaces at the tips of the teeth.
 12. The apparatus of claim9, wherein the wedge-shaped teeth are configured to create thewedge-shaped sample wells with larger openings to the wedge-shapedsample wells at the at the top of the wedge-shaped sample wells.
 13. Theapparatus of claim 9, wherein the wedge-shaped teeth have a top that isbetween about 0.15 inch and about 0.2 inch in depth.
 14. The apparatusof claim 9, wherein the edge of the comb opposite to the wedge-shapedteeth serves as a handle to facilitate insertion of the comb into thecassette and removal of the comb from the cassette.
 15. The apparatus ofclaim 9, wherein the wedge-shaped teeth have at least one surfaceextending from a plane parallel to the bottom surface of the teeth atangle of 70 degrees.
 16. The apparatus of claim 1, wherein the apparatusis configured to create a gel having sharp bands or a gel having highresolution.
 17. The apparatus of claim 1, wherein the cassette has atleast one interior surface.
 18. The apparatus of claim 17, wherein theat least one interior surface is coated with a coating.
 19. Theapparatus of claim 18, wherein the coating is an oxygen interferencecoating.
 20. The apparatus of claim 19, wherein the oxygen interferencecoating is selected from at least one of SiO or SiO₂.
 21. An apparatusfor gel electrophoresis comprising a comb having wedge-shaped teethconfigured to create wedge-shaped sample wells in a gel by insertion ofthe comb into the gel and by removal of the comb once the gel has beencast, wherein the wedge-shaped teeth have at least one surface extendingfrom a plane at the bottom surface of the teeth at an angle of between45 degrees and 90 degrees to the top of the teeth, wherein the combfurther comprises a recessed area on the back of the comb where the combcontacts a gel cassette.